The Salicaceae family is of scientific interest for both biomass production and for the study of sex chromosome evolution and sex determination in plants. Salicaceae includes two genera of economic importance: Populus and Salix, whose native habitats extend to every inhabited continent, except Australia. However, the family is almost entirely dioecious, with the sex determination regions (SDR) localized to different chromosomes and exhibiting different heterogametic systems across taxa. As a result, there is substantial interest in understanding the precise mechanisms of sex determination within this family and how they vary between species, as this will provide clues to the evolution of dioecy. Moreover, dioecy presents several barriers to the development of shrub willow as a bioenergy crop by limiting possible breeding combinations between males and females, hindering inbred line development, and confounding the selection of several correlated biomass traits, such as crown diameter. This research seeks to understand the genetic mechanisms responsible for sex determination in the shrub willows, in order to enable more effective breeding strategies and genetics studies. The primary objectives of this study were to: 1) evaluate sex dimorphism in floral tissue across a Salix purpurea F2 family and identify candidate sex determination genes; 2) characterize the genetic basis of monoecy in a monoecious Salix purpurea and its progeny and refine hypotheses for the mechanisms of sex determination in this species; 3) perform functional genomic analysis of candidate Salix sex determination genes; 4) present annotations of 11 genomes from six shrub willow species and characterize their respective SDRs, and 5) explore future functional genomics opportunities in S. purpurea through protoplast transformation and CRISPR gene editing. The results from this work provide evidence to support a two-gene system of sex determination in S. purpurea mediated by ARR17 and GATA15 that is different from the related genus Populus. This study also presents a variety of new resources for willow breeding and genomics including the release of 11 new annotated genomes, monoecious willows for use in inbred line development and genetic mapping studies, and protoplast transformation as a tool for Salix functional genomics in vivo.